Co-integrate Col3m bla NDM-1-harboring plasmids in clinical Providencia rettgeri isolates from Argentina

ABSTRACT The first cases of bla NDM in Argentina were detected in three Providencia rettgeri (Pre) recovered from two hospitals in Buenos Aires city in 2013. The isolates were genetically related, but the plasmid profile was different. Here, we characterized the bla NDM-1-harboring plasmids of the first three cases detected in Argentina. Hybrid assembly obtained from short- and long-read sequencing rendered bla NDM-1 in Col3M plasmids of ca. 320 kb (p15268A_320) in isolate PreM15268, 210 kb (p15758B_210) in PreM15758, and 225 kb (p15973A_225) in PreM15973. In addition, PreM15758 harbored a 98-kb circular plasmid (p15758C_98) flanked by a putative recombination site (hin-TnAs2), with 100% nucleotide ID and coverage with p15628A_320. Analysis of PFGE/S1-nuclease gel, Southern hybridization with bla NDM-1 probe, hybrid assembly of short and long reads suggests that pM15758C_98 can integrate by homologous recombination. The three bla NDM-1-plasmids were non-conjugative in vitro. Moreover, tra genes were incomplete, and oriT was not found in the three bla NDM-1-plasmids. In two isolates, blaNDM-1 was embedded in a partially conserved structure flanked by two ISKox2. In addition, all plasmids harbored aph(3')-Ia, aph(3')-VI, and qnrD1 genes and aac(6´)Ib-cr, bla OXA-1, catB3, and arr3 as part of a class 1 integron. Also, p15268A_320 and p15973A_225 harbored bla PER-2. To the best of our knowledge, this is the first report of clinical P. rettgeri harboring blaNDM-1 in an atypical genetic environment and located in unusual chimeric Col3M plasmids. The study and continuous surveillance of these pathogens are crucial to tracking the evolution of these resistant plasmids and finding solutions to tackle their dissemination. IMPORTANCE Infections caused by carbapenem hydrolyzing enzymes like NDM (New Delhi metallo-beta-lactamase) represent a serious problem worldwide because they restrict available treatment options and increase morbidity and mortality, and treatment failure prolongs hospital stays. The first three cases of NDM in Argentina were caused by genetically related P. rettgeri recovered in two hospitals. In this work, we studied the genetic structure of the plasmids encoding bla NDM in those index cases and revealed the enormous plasticity of these genetic elements. In particular, we found a small plasmid that was also found inserted in the larger plasmids by homologous recombination as a co-integrate element. We also found that the bla NDM plasmids were not able to transfer or move to other hosts, suggesting their role as reservoir elements for the acquisition of resistance genes. It is necessary to unravel the dissemination strategies and the evolution of these resistant plasmids to find solutions to tackle their spread.


Introduction
-and includes -and bacteremia -like ampicillin -first-generation -Please check reference 6 concerning the mentioned genes.
-has been widely -an epidemiological link -ionization-time of -azide-resistant 2. Materials and Methods 2.1 Clinical isolates -reference 11 is missing in the text -table S1 presents epidemiological information and antibiotics MIC not primers list -per the manufacturer's instructions.
-the quality of the DNA library.
-and elution was carried out -split files and trim barcodes -ResFinder and PlasmidFinder -was analyzed by calculating a pairwise Results -phenotypic profile as PreM15268 and -the text mentioned all isolates were resistant to aztreonam, but in table S1 strain PreM15758 is susceptible.
-The quality of figure 2 is not adequate and makes it impossible to analyze the alignment figure .-Is not clear the comment concerning p15758A_312, since this plasmid is only mentioned in line 196 and no data is available in table 1.
-integrated into an IncC plasmid -it is mentioned that all isolates are susceptible to amikacin, even though the three of them harbored aac(6´)Ib-cr and aph genes.Discussion -known to act as a DNA Reviewer #2 (Comments for the Author): The article describes the genome of the first three NDM-1 producing P. rettgeri strains isolated in Argentina.In addition to the detailed study of the plasmids that carry the blaNDM-1 gene, a very interesting homologous recombination event is also described in this clone, which results in distinct Col3M plasmids.The genetic environment that surrounds the blaNDM-1 gene is not the most common one found in Latin America, but it was recently described in Uruguay and its dissemination deserves attention.Below are minor corrections: Line 39: Morganellaceae Line 87: were, instead of was Line 97: ...to determine the quality... Line 114: and and Line 119: I guess there is a comma instead of a period after v.0.95Line 129: ...similar or identical... Line 139: was, instead of were Lines 189-190: "Of note, blaPER-2 was only found in p15628A_320."Actually, it was also found in p15793A_225.Maybe you mean that between the index strains, it was only found in p15628A_320.Please clarify the sentence.Line 253: as as

Staff Comments:
Preparing Revision Guidelines To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to -table S1 presents epidemiological information and antibiotics MIC not primers list Accepted, Thank you for the observation.The primer list was added to the supplementary material as Table S1 and the table containing the epidemiological data was re named to Table S2.
-per the manufacturer's instructions.Accepted -the quality of the DNA library.Accepted -Is not clear the comment concerning p15758A_312, since this plasmid is only mentioned in line 196 and no data is available in table 1.
Thank you for your comment.We mentioned p15758A_312 because PreM15758 showed a 312 Kb band seen in the S1-PFGE gel with positive hybridization using the bla NDM probe.
When it was sequenced, we did not find a 312 Kb plasmid.This is why Table 1 only has the 312 bold number (explained in the table 1 footnote) and no additional information.Moreover, we mention p15758A_312 in Lines 198 -201 to explain / hypothesize the fact that p15758C_98 may be "inserting" or "excising" from the larger plasmid by homologous recombination.We have now modified the table's footnote and added details to make it clearer and minimize confusion.
-integrated into an IncC plasmid Accepted.
-it is mentioned that all isolates are susceptible to amikacin, even though the three of them harbored aac(6´)Ib-cr and aph genes.

REVIEWER #2
We would like to thank reviewer #2 for the constructive revision of our manuscript.We have accepted and answered all the queries raised.Our responses can be found below each comment Reviewer #2 (Comments for the Author): The article describes the genome of the first three NDM-1 producing P. rettgeri strains isolated in Argentina.In addition to the detailed study of the plasmids that carry the blaNDM-1 gene, a very interesting homologous recombination event is also described in this clone, which results in distinct Col3M plasmids.The genetic environment that surrounds the blaNDM-1 gene is not the most common one found in Latin America, but it was recently described in Uruguay and its dissemination deserves attention.Below are minor corrections: Line 39: Morganellaceae Accepted Line 87: were, instead of was Accepted Line 97: ...to determine the quality... Accepted Line 114: and and Accepted Line 119: I guess there is a comma instead of a period after v.0.95Accepted Line 129: ...similar or identical... Accepted Line 139: was, instead of were Accepted Lines 189-190: "Of note, blaPER-2 was only found in p15628A_320."Actually, it was also found in p15793A_225.Maybe you mean that between the index strains, it was only found in p15628A_320.Please clarify the sentence.Congratulation!!Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.

Accepted
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Please check reference 6 concerning the mentioned genes.Accepted.Thank you for your observation.The reference was changed accordingly -has been widely Accepted -an epidemiological link Accepted -ionization-time of Accepted -azide-resistant Accepted 2. Materials and Methods 2.1 Clinical isolates -reference 11 is missing in the text Accepted.The mistake was fixed.

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and elution was carried out Accepted -split files and trim barcodes Accepted -ResFinder and PlasmidFinder Accepted -was analyzed by calculating a pairwise Accepted Results -phenotypic profile as PreM15268 and Accepted -the text mentioned all isolates were resistant to aztreonam, but in table S1 strain PreM15758 is susceptible.Accepted.Thank you for the observation.The sentence was modified accordingly -The quality of figure 2 is not adequate and makes it impossible to analyze the alignment figure.Accepted.Thank you for the observation.I replaced figure 1 and figure 2 by the tif 600 dpi images.
Yes, in fact the three isolates are susceptible to gentamicin and amikacin.The susceptibility to certain aminoglycosides in Providencia spp harboring these genes has been documented in several articles, although, to the best of our knowledge, the reason has not been explained.In particular, susceptibility to amikacin by P. rettgeri strains was reported by Ingo Stock and B. Wiedemann in "Natural antibiotic susceptibility of Providencia stuartii, P. rettgeri, P. alcalifaciens and P. rustigianii strains Antimicrobial susceptibility" J. Med.Microbiol.-Vol.47 (1 998), 629-642.In the case of aac(6´)Ib-cr, this modifying enzyme mostly affects ciprofloxacin and norfloxacin by acetylation, but the inactivation effect over gentamicin and amikacin is either poor or variable.In our experience, aac(6´)Ib-cr does not affect gentamicin and amikacin, as reported previously (see Table 2 in Andrés, P. et. al. Antimicrobial Agents and Chemotherapy, 2013, 57 (6), p. 2467-2475.).In our epidemiology, high level resistance to amikacin and gentamicin is mostly seen when rmt enzymes are present inserted in the genetic element with bla NDM (usually in the A/C plasmid), or with aac(6´)-Ib, which confers resistance to gentamicin and amikacin.